Recent studies of our group showed that partial resistance against E. caproni secondary infections is developed after chemotherapeutic cure of a primary infection and innately produced IL-25 is crucial to determine the resistance. Susceptibility to primary infections was associated with low levels of intestinal IL-25 expression, whilst deworming via administration of praziquantel was accompanied by a steady increase in IL-25 expression that prevented the establishment of secondary infections [14–15]. However, it is not well defined if the participation of IL-25 in resistance to infection. Herein, we analyze the proteomic changes indiced by IL-25 that may contribute to resistance to infection.
Resistance against E. caproni infection has been associated with the preservation of the intestinal homeostasis despite the possible damage induced by the parasite. In resistant hosts, E. caproni infection elicits a rapid renewal of the intestinal that allows to maintain the epithelium homeostasis and impairing the proper worm establishment. In contrast, in susceptible hosts, such as mice, the development of chronic infections are related to the disruption of the intestinal homeostasis causing tissue hyperplasia [19–21]. Although mice is a susceptible host, treatment with rIL-25 prior to infection induces complete resistance to the infection . Our results support that IL-25 may contribute to resistance by the enhancement of intestinal homeostasis via activation of the canonical wingless-related integrator site (Wnt)/β-Catenin signaling pathway. Treatment of naïve mice with rIL-25 only elicited changes in the production of a total of 5 proteins, including the structural protein junction plakoglobin or γ-catenin. This protein is a member of the catenin family, paralog to β-catenin, and is a component of desmosomes. It is involved in the mechanisms of cell adhesion and is essential to maintain and regulate intestinal epithelial homeostasis [22–24]. Plakoglobin participates in the canonical pathway of Wnt/β-Catenin signaling since elevated levels of plakoglin promote the stabilization and nuclear localization of β-catenin enhancing the activation of Wnt/β-Catenin signaling . Activation of this pathway is essential for the maintenance of the intestinal homeostasis since it plays an essential role in regulating cell proliferation, survival, and differentiation facilitating epithelial healing after disruption . The central mediator of Wnt signaling is β-catenin. Wnt signaling activation is dependent on the nuclear translocation of β-catenin. When a canonical Wnt binds to the frizzled receptor and its co-receptor lipoprotein receptor-related protein 5/6, dishevelled is recruited and the destruction complex is inhibited, thus promoting the accumulation of non-phosphorylated β-catenin in the cytosol. As non-phosphorylated β-catenin accumulates intracellularly, it is translocated to the nucleus where it activates the T-cell factor/lymphocyte enhancer factor transcription factor families to regulate gene transcription . Plakoglobin participates in the canonical pathway of Wnt/β-Catenin signaling since this protein inhibits the glycogen synthase kinase (GSK3β)-mediated nuclear localization of β-catenin. GSK-3β is one important member that regulates the Wnt/β-catenin target gene expression by controlling the level of cytoplasmic β-catenin and its nuclear shuttle . Elevated levels of plakoglin promote the stabilization and nuclear localization of β-catenin  and may enhance intestinal homeostasis despite the damage caused by the infection. Oudhoff and co-workers  reported that Wnt/β-Catenin signaling is an important component of resistance to the intestinal nematode Trichuris muris in mice. These authors showed that Wnt expression programs are induced upon infection with T. muris eggs and wild type mice were able to expel the infection. In contrast, mice deficient in SETD7 (a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax domain-containing family of lysine methyltransferases) were not able to reject the infection. SETD7 controls IEC turn over by modulating developmental signaling pathway Wnt/β-Catenin. Lack of SETD7 resulted in downregulation of Wnt/β-catenin, deficient and susceptibility to infection . The fact exposure of rIL-25-treated mice to E. caproni metacercariae induced a significant downregulation of three isoforms of plakoglobin with respect to rIL-25-treated mice supports that plakoglobin plays an important role in E. caproni infections and its potential role in the development of resistance to infection.
Strikingly, two other proteins involved in cell differentiation and tissue homeostasis also became altered by the treatment with rIL-25. Proliferation-associated 2G4 [PA2G4) and receptor of activated protein C kinase 1 (RACK1) were found to be downregulated in rIL-25-treated mice with respect to naïve mice. PA2G4, also known as EBP1, is a RNA-binding protein that is involved in growth regulation. This protein is present in pre-ribosomal ribonucleoprotein complexes and may be involved in ribosome assembly and the regulation of intermediate and late steps of rRNA processing. This protein can interact with the cytoplasmic domain of the ErbB3 receptor and may contribute to transducing growth regulatory signals. This protein is also a transcriptional corepressor of androgen receptor-regulated genes and other cell cycle regulatory genes through its interactions with histone deacetylases. This protein has been implicated in growth inhibition [30–31]. The EBP1-binding in promoters regulated by E2F can result in an enhanced ability of EBP1 to suppress genes transcription regulated by the cell cycle and inhibit cell growth [30, 32]. Furthermore, the expression of EBP1 generates the negative expression of the androgen receptor (AR) and several of its target genes, thereby inhibiting AR-regulated cell growth [30–33]. RACK1 is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins (Gβ). RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilizing protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. RACK1 is a scaffolding protein that takes part in the maintenance of intestinal homeostasis protecting the integrity of the epithelial barrier by suppressing the regeneration and proliferation of crypt cells, promotes differentiation and apoptosis and is generated against stress responses [34–36]. Downregulation of both EBP1 and RACK1 may contribute to prevent the hyperplasia in the intestinal tissue that is associated to susceptibility to E. caproni infections.
Another striking feature that may be related with alterations in the intestinal epithelium and resistance to infections is in the upregulation of annexins 2 and 4 min rIL-25-treated mice exposed to E. caproni metacercariae.. Annexin is a common name for a family of structurally related proteins that mostly found in eukaryotic organisms both in extra and intracellular environment and bind phospholipids and carbohydrates in the presence of Ca2+ [37–38]. Annexins play a role in the control of cell death and affect membrane properties such as permeability or anchoring of cytoskeletal elements [39–40]. These proteins also are related to epithelial cell migration that is a critical event in gastrointestinal mucosal wound healing  Furthermore, evidences of annexins as modulators of inflamation have been widely provided . In the small intestine, the expression of annexins appears to be restricted to M cells, where it plays a role in endocytic transport and membrane scaffolding . Annexins can function as a natural ligand for phosphatidylserine, a prominent phospholipid that is exposed during cell death. It has been suggested that annexins blocks posphatidylserine-dependent phagocytosis of dying cells, forcing its internalization and delivering phosphatidylserine back to the inner leaflet of the cell membrane . Annexins have been implicated in the repair mechanisms on both tissue and intracelular levels . Upregulation of annexins has been reported in association with resistance to E. caproni secondary infections in mice . This was attributed to the reduced rate of cell death that happens despite induction of mitochondrial dysfunction, cellular senescence and high levels of oxidative stress .
Specifically, annexin 4 appears to play a specific role in membrane repair. Plasma membrane repair mechanisms involve internalization via endocitosis, or exocytosis as observed from mechanical wounding or exposure to plasma membrane poreforming agents [45–48]. Therefore, overexpression of annexin 4 due to the exposurte to metacercariae of rIL-25-treated mice may contribute to the defense of this parasite infection participating in the healing of the intestinal tissue and acting as an anti-inflammatory factor. Annexin 2 is a protein that is part of the lipid rafts in the intestinal brush border and is associated with actin filaments mediating in membrane-membrane and membrane-cytoskeletal interactions influencing actin cytoskeletal remodeling through targeting signaling molecules to membrane domains. As a consequence it plays an important role in membrane trafficking and stabilization of membrane-associated protein complexes with the actin cytoskeleton and has been implicated in the migration of various cell types including epithelial cells and cell matrix interaction [41, 49]. Moreover, annexin 2 has been shown to induce clustering of specific plasma membrane phospholipids and play a role in lipid domain formation . The absence of annexin 2 would therefore influence RhoA-mediated F-actin reorganization, which in turn affects motility of annexin 2 defficient cells . In this sense, our results suggest that the up-regulation of both annexins (annexin 2 and 4) could help maintain the epithelial barrier structure during helminth infections.
Quantitatively, the proteins involved in metabolic processes were the most altered in any of the groups studied. A generalized reduction of ileal cell metabolism has been observed at 2 weeks after E. caproni infection in presence of rIL-25. A total of twenty of the identified spots (corresponding to 15 different proteins) are metabolic enzymes and a great part of them were significantly downregulate in infected in presence of rIL-25 mice with respect to control inoculated with rIL-25. Alterations in several proteins involved in the Krebs cycle (fumarate hydratase and malate dehydrogenase) and in the pentose phosphate pathway (transaldolase and 6-phosphogluconate dehydrogenase). We also detected a reduced expression of several glycolytic enzymes including several isoforms of enolase 1B, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase PKM, phosphoglycerate kinase 1 and triosephosphate isomerase. This may indicate mitochondrial dysfunction and a subsequent decrease in aerobic metabolism as a consequence of the exposure to E. caproni metacercariae. A similar situation has been described in the ileum of E. caproni mice at 2 wppi . The reduction of aerobic metabolism was associated with an increase in the anaerobic use of glucose, through the overexpression of lactate dehydrogenase. However, Cortés and co-workers  detected a marked downregulation of the production of lactate dehydrogenase were in the ileum of resistant secondarily infected mice, suggesting that both aerobic and anaerobic metabolism become impaired as the infection progresses. In contrast, in our study, lactate dehydrogenase was upregulated in the ileum of rIL-25-treated mice exposed to the infection with respect to mice conventionally infected. This might indicate that infection requires an increase in the anaerobic use of glucose to supports the high energy demand caused by parasitic infection both presence/absence of rIL-25 to cover the metabolic demand generated by mitochondrial dysfunction. The impact that alterations in energy metabolism has over the course of the infection is difficult to assess according to our current knowledge. However, it could be of relevance for a better understanding of the mechanisms developed in the intestinal environment in responses to helminth infections.
Several antioxidant and detoxifying enzymes such as peroxiredoxins 1 and 4, glutathione S-transferase and dihydropteridine reductase were also found to be altered. Treatment with rIL-25 induced a marked downregulation of peroxiredoxin 4. This enzyme is a ubiquitously expressed member of the peroxiredoxin family that is localized in the endoplasmic reticulum and extracellular space . Peroxiredoxin 4 diminishes oxidative stress by reducing hydrogen peroxide to water in a thiol-dependent catalytic cycle and has been linked to the regulation of the key pro-inflammatory transcription factor, nuclear factor kappa B (NF-κB) [53–55]. This supports that the processes related to oxidative stress and cell death are altered in the presence of infection by E. caproni independently of the presence and IL-25. IL-25 does not appear to take part in the regulation of the processes related to oxidative stress and apoptosis necessary to maintain intestinal homeostasis. Strikingly, exposure of rIL-25-treated mice to metacercariae caused a downregulation of peroxiredoxin 1 instead peroxiredoxin 4. Peroxiredoxin 1 plays a key role against reactive oxygen species and antioxidants and in in inflammatory responses . The production of this enzyme is upregulated in active ulcerative colitis specimens, and it increases along with the inflammation level in ulcerative colitis regenerative mucosal crypt epithelial cells [57–58]. Downregulation of peroxiredoxin 1 was observed as a consequence of the curation of an E. caproni infection . The reduced production of this enzyme after infection in presence of rIL-25 may play a double role, promoting crypt-cell proliferation but, at the same time, inducing oxidative stress and ROS-mediated programmed cell death to counteract homeostatic dysregulation induced by the infection [21, 59–60].
Infection of rIL-25-treated mice also induced reduction in the production of palmitoyl-protein thiosterase (PPT). Protein thioestherases, or depalmitoylases, mediate the depalmitoylation of modified proteins, thereby completing a cycle of this reversible post-translational modification [61–63]. Palmitoylation can effectively act as a post-translational “switch” on some proteins and provide dynamic control over protein localization or function. Indeed, palmitoylation plays critical roles in protein trafficking and strongly influences the stability of proteins [64–69]. PPT1 is a lysosomal substrate that enter in the lysosome via autophagy leading to signaling of several processes related with anabolic and catabolic metabolism in the cell [63, 70]. PTT deficiency has been implicated in the disruption of the lysosome-endosomal pathway and in other cellular processes, including endocytosis, vesicular trafficking, synaptic function, lipid metabolism, neural specification, and axon connectivity and seems to be involved in cell susceptibility to apoptotic cell death and defects in the mitochondrial enzyme activities and adaptive energy metabolism . For this reason, Downregulation of PPT after exposure to E. caproni metacercariae presence of rIL-25 mice may be due to its role in processses regulation involved in cell death and energy metabolism in order to maintain intestinal homeostasis. This is supported by the concomitant downregulation of creatine kinase B-type (CKB). This enzyme plays a critical role in energy transduction in tissues with increases in energy demands. The creatine kinase energy system is itself further regulated by hypoxic signaling and can enhance creatine metabolism during oxygen deprivation to promote tissue healing and homeostasis . Impaired Cr/PCr shuttling may contribute to dysregulated mitochondrial energetics and an increased barrier permeability characteristic of inflamed mucosa ans susceptibility to E. caproni infection [9, 21, 73].